Grunstein and Hogness, Proc. Natal. Acad. Sci. USA, 72, 3961-3965 (1975), described in 1975 a colony hybridization method which enabled them to screen for hybrid plasmids containing specified DNA sequences of genes. They lysed colonies in situ on nitrocellulose papers and fixed their denatured DNA, which was subsequently hybridized to specific labelled RNA and visualized by autoradiography. They were dealing with plasmid derivatives of ColEl, which would be present in large numbers (&lt;10 per chromosome) and therefore their method was sufficiently sensitive for their purposes.
Gergen et al., Nucleic Acid Res., 7, 2115-2136 (1979), subsequently described a simpler and cheaper method, which involved lysing the cells on Whatman-brand 541 filter paper (a high wet strength paper). The DNA seemed to be immobilized very rapidly on this paper so that they were able to treat the filters in batch form without any special precautions. However, these investigators were also dealing with a multi-copy amplifiable plasmid, and they indeed amplified the plasmid by a chloramphenicol incubation step before lysing the colonies. An advantage of the 541 paper is that no baking of the papers is required to fix the DNA.
Kaper et al., Infec. Immun. 32, 661-667 (1981), adapted to colony hybridization method of Grunstein and Hogness to probe for the presence of cholera enterotoxin genes. Mosely et al., J. Infect. Dis., 145, 863-869 (1982), used this method to identify enterotoxigenic E. Coli strains by using three different enterotoxin gene probes. However, attempts by the present inventor to identify enterotoxigenic strains isolated from patients with diarrhea in Sao Paulo, Brazil have revealed that there are difficulties with heat-stable enterotoxin (ST) probes. The signal is often weak, and when compensated by exposing the films for longer time periods, the negative controls also gave a positive response.